The long-term objective of the proposed work is to use invasive tumors of Drosophila as a model system to investigate the cascade of events that begins with loss of function of tumor suppressor genes and culminates in the altered expression of effector genes which mediate invasiveness such as gelatinases and their inhibitors. Seven Drosophila tumor suppressor genes have been identified by mutations which cause overgrowth of larval brains. Mutations in the best studied gene of this type, lethal giant larvae, cause loss of normal tissue structure and loss of capacity to differentiate of imaginal neuroblasts. The invasiveness of neuroblastoma cells derived from such mutant brains following transplantation into adult hosts has been confirmed using a cell-autonomous marker. One aim of this proposal is to use this cell marking method to analyze the invasive potential of Drosophila neuroblastomas caused by mutations in the six other tumor suppressor genes. This analysis will include a qualitative study of the host tissue specific susceptibility to invasion and a quantitative comparison of the degree of invasiveness. Two approaches are planned to investigate the cascade of events that begins with loss of function of tumor suppressor genes. One is to examine the genetic hierarchy of the seven already identified genes. The other is to use a temperature sensitive mutation in the malignant brain tumor gene to screen for second site mutations that identify downstream genes whose function is required to express the invasive phenotype. The metastatic phenotype includes loss of normal tissue integrity and increased proteolysis of the extracellular matrix. Either or both of these processes can be mediated by gelatinases. Preliminary work has led to the detection of three Drosophila proteins with gelatinase activity and demonstrated increased accumulation of one of these three in brains dissected from lethal giant larvae mutants and in invasive neuroblastomas derived from such mutant brains. This gelatinase is a 49kDa protein with a pI of 7.2 that cross-reacts with antibody directed against human gelatinase A. The neuroblastomas caused by mutations in the six other genes will be screened for increased accumulation of this protein as well as the two other gelatinases. The gelatinase 7.2 gene will be cloned and mutations will be recovered in it. The consequences of these gelatinase mutations on the invasive phenotype caused by mutations in the seven tumor suppressor mutations will be examined. These mutations will be used as starting points to identify upstream genes that regulate expression of the gelatinase 7.2 gene in tumors. The decision to focus initially on gelatinase 7.2 has been dictated by preliminary results, by the availability of immunological reagents, and by progress already made towards cloning of the gelatinase 7.2 gene. Preliminary work has also shown evidence of putative inhibitors of gelatinase in Drosophila larvae. Ultimately, a similar approach will be applied to the study of the two other gelatinases and these inhibitors.